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Proteolytic enzyme activity of colloid extracted from single follicles of the rat thyroid.

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PROTEOLYTIC ENZYME ACTIVITY O F COLLOID
EXTRACTED FROM SINGLE FOLLICLES
O F THE RAT THYROID1
E. DE ROBERTIS *
Departmelit of Anatomy, Johns Hopkins University, BalthWTe, MaryEand and
Institute of General Anatomy and Embryology, University
of Bucnos Aires, Argentine
ONE PLATE (FIVE FIQURES)
Knowledge of the properties and composition of thyroid
colloid is fragmentary. It was thought that studies of colloid
extracted from single follicles of the thyroid gland would
help to fill this gap. Experiments of this sort were performed
with microdissection techniques on the thyroid gland of living
rats. As a result it becomes possible to reinterpret the appearance of the colloid in fixed preparations in the light
of its state in the living animal. I n addition, the results support earlier evidence that colloid may be reabsorbed from
the follicles by means of the previous action of a proteolytic
enzyme present in this fluid. This enzyme is thus a second
known component of colloid, the other being thyroglobulin.
According to this concept, thyroglobulin is hydrolized in
the lumen of the follicle and the smaller molecules resulting
from this hydrolysis become available for transport through
the follicular cells. It was determined that there are fluctuations in the extent of proteolytic activity not only in different follicles of normal glands but also (to a greater extent)
under certain experimental conditions : i.e., after the administration of potassium iodide and of thyrotropic pituitary
This research is part of a project supported by an allotment from the Rockefeller Foundation Fluid Research Fund.
Fellow of the Rockefeller Foundation.
219
220
E. DE ROBERT18
extract. Estimates of the p H of the colloid fluid served to
show that such changes in enzymatic activity seem to be
independent of this factor.
METHOD
Rats were anesthetized with nembutal, supplemented occasionally with ether. The thyroid gland was exposed, and
the isthmus illuminated from above I n this portion of the
gland the follicles are disposed in few layers deep, so that
micromanipulation is simplified. Colloid was aspirated sometimes from lobar follicles. Micropipettes with a bore of 5-20 CI
were used. Contamination by tissue fluid was prevented by
slight drying of the surface (as recommended by Wearn and
Richards, '26'25) and by filling the pipette to the tip with
mercury.
The aspirated colloid was diluted in the micropipette with
five to ten times its volume of buffer and tested for the presence
of proteolytic activity by the method recommended by Pickford
and Dorris ( '34). Lantern slide plates are cleared of silver
salts, washed in running water, dried and cut into thin strips.
The buffered colloid, which forms a droplet of the order of
0.5 mm. in diameter, is carefully deposited on the gelatin film
under the binocular microscope. The plate is transferred
rapidly to a moist chamber where it is kept at 2625°C. for
2 hours. After this time the slides are rinsed with distilled
water followed by washing in running water for several hours.
This procedure stops digestion and effects a uniform swelling
of the gelatin film. The slides while wet were stained with
1% acid fuchsin for 5 minutes, rinsed in distilled water and
95% alcohol, and then dried. Observations of the degree of
digestion were made with low magnifications of the microscope.
The degree of digestion was judged by the depth of staining
in the crater formed by the sample droplet. Control digestions with buffer alone yielded negative results. Confirming
Pickford and Dorris, it was found that Pancreatin Merck
produces strongly positive digestion in a dilution of 1 :1000 ;
the reaction is positive in a dilution of 1: 5000.
PROTEOLYTIC ENZYME ACTIVITY O F COLLOID
221
As these investigators pointed out, this micromethod is not
quantitative. I n addition, it is very difficult to aspirate exactly
the same amount of colloid to be diluted with the same volume
of buffer solution from different follicles. Nevertheless, we believe that it is possible to make a semiquantitative estimation
of changes in enzymatic activity, The results were estimated
as negative (-), faintly positive (+), positive (+),strongly
positive (++),and total digestion (+++).
A relative estimate of the viscosity of colloid was made
on the basis of the ease or difficulty in aspirating and expelling
the colloid from the pipette.
I n order to determine the p H of the colloid, indicators of
the Clark and Lubs series were injected into the follicle with
a small-bore pipette. Injections were also made in the perifollicular connective tissue. In some cases the time of diffusion
of the indicator within the follicle, and of its subsequent disappearance, was measured. The indicators were made up in
saturated aqueous solution. This seemed a justifiable way of
increasing color density because the size of the droplet injected
was so minute as compared with the volume of colloid in the
follicle.
OBSERVATIONS
Colloid and erzxyme activity in normal rats. The colloid
aspirated from follicles of normal rats is very viscous: that
is, it rises in the micropipette very slowly and with a high
negative pressure. It is quite brilliant and always unquestionably fluid. Sometimes the droplets appear faintly yellow
with reflected light, but generally they are colorless. The
colloid is optically homogeneous and its viscosity in different
parts of single follicles seems to be uniform. No vacuoles
were observed at any time in the colloid even on observation
with high powers of the microscope. Contamination with blood
cells from the perifollicular capillaries was observed in very
few cases.; in these, the erythrocytes were easily visible with
the binocular microscope. Vascular injury seems to be minimized by closure of the perifollicular capillaries as a result
of compression by the pipette.
222
E. DE ROBERTIS
The proteolytic activity of colloid from different individual
follicles of normal rat glands is shown in table 1. It was
tested in the “physiological range” of pH, between 7.4 and
6.6. I n all cases the reaction was positive but not very marked.
These results were checked with the buffer solution alone
and by denaturing the protein with formalin and mercuric
chloride. I n these three latter procedures the results were
TABLE 1
Proteolytic activity of colloid in normal arid experinimtol animals
Norinal rats
+
+
Rats treated with uotassium iodide
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
+
+++
+
++, ++
++
++
+, *
93
70
90
90
100
83
93
11 days
12 days
45 days
-+
,-r-
*,rt
93
I
1
1
1
’
++,+,+
++,++
105
100
107
125
107
80
305
125
93
+-I-+,+,+
+, ++
+
+
+, +
f,
c
++,-if
++
1 ++,
++
+,++,++
1
’
++,+-I-
I +,+,+
5 hours
6 hours
10 hours
16 hours
20 hours
24 hours
28 hours
44 hours
48 hours
10
13
10
10
20
10
15
1.5
20
PROTEOLYTIC ENZYME ACTIVITY OF C O U O I D
223
negative. I n rat 4 of table 1, colloid was permitted to act
on the gelatin film for +,1 and 2 hours.
Action of pot,cassium iodide on the activity of the proteolytic
elzzyme. Rats fed a normal diet with 1% of potassium iodide
added to the ground food showed a marked change in the
properties of the colloid and in the activity of the proteolytic
enzyme (see table 1). The results differ according to the
time during which potassium iodide was administered. One
group of six rats was so fed for a period of 5 to 12 days ; the
second group of four rats was treated for 45 to 54 days. I n
the first group, vascularity was improved, the colloid was
less viscous, and the activity of the enzyme was greatly increased (table 1). I n the second group the colloid was highly
viscous; in fact, it seemed sometimes more viscous than in
normal glands. Coincidentally, the activity of the enzyme
was decreased below the normal level (table 1).
Activity of the ewxyme after ilzjectiow of pituitary eatract
colztaimilzg thyrotropic fact,or. Rats were injected intraperitoneally with doses of thyrotropic pituitary extract ranging
between 1and 2 mg. of dry weight (1mg. contained 10 guinea
pig units) in one or two injections. Animals were studied
between 5 and 48 hours after the first injection.
The follicles in all cases showed increased vascularity with
marked dilatation of the perifollicular capillaries. The viscosity of the colloid was greatly decreased: it was very easily
aspirated in the micropipette. The refractive index seemed
to be diminished: I n general, the activity of the proteolytic
enzyme was increased especially when the colloid was taken
a short time after the injection (5-24 hours) (table 1).I n these
cases, the results generally were strongly positive.
Nevertheless, in two cases when colloid was aspirated a
longer time after the administration of the pituitary extract
(44-48 hours), the activity fell within the limits of the normal
animal. No record of the viscosity of the fluid were made in
these two rats.
Effect of the p H of the buffer on the activity of the enzyme.
Several scattered observations are summarized in table 2. The
224
E. DE ROBERTIS
general trend is f o r the activity of the enzyme to increase
greatly in the acid range, especially at pH 3 and pH 2. At
pH 8.2, the activity seems to be lower than within the physiological range. This variation of enzymatic activity with pH
is seen in the colloid of normal and also experimental rats.
- negative, & faintly positive,
digestion.
+ positive, ++ strongly positive, +++ total
The pH of the imtrafolliczclar colloid and of the perifolliczclar
tissue. Table 3 gives a general idea of the results found in
follicles of more than twenty normal and experimental rats.
Four indicators with overlapping p K were injected : (chlorphenol red, brom-cresol purple, brom-thymol blue, and phenol
red).
Brom-thymol blue gave the most striking results. When
injected into the follicle its color is yellow-green or green
PROTEOLYTIC ENZYME ACTIVITY OF COLLOID
225
( p H 6.5-6.7) and when injected outside of the follicle the
color is blue ( p H 7.1). The other indicators checked with
these results in all cases. The use of electrometric methods
for the determination of the pH of colloid would give more
accurate results. Nevertheless, the fact that the indicators
used overlap in their effective ranges permit of some confidence in the determinations reported in this paper.
While the indicator is injected into the colloid it spreads
through the whole follicle and reaches the periphery in 30
to 90 seconds, after which it appears optically uniform in
density. Thereafter, the color intensity decreases regularly
and uniformly over the whole follicle and after 3 minutes 30
CPR, ohlor-phenol red ; BCP, brom-cresol purple ; BTB, brom-thyntol blue ;
PR, phenol red.
seconds to 15 minutes the indicator disappears from the follicle. This is probably due to the diffusion of the indicator
through the follicular wall. When it is injected just before
the death of the animal it remains unchanged in the follicle
for a t least 30 minutes. The indicator injected in the perifollicular connective tissue of the living gland with good
circulation also diffuses, and disappears very rapidly, in
about 1or 2 minutes.
DISCUSSION AND SUMMARY
The application of microdissection methods to the thyroid
gland of the rat has resulted in more direct information concerning the colloid. Colloid appears in the follicles of normal
226
E. DE ROBERTIS
animals as a highly viscous but fluid substance with high
refractility. The fact that the indicator injected in the follicles
spreads rapidly from the point of injection throughout the
whole follicle, also is a confirmation of the fluidity of the
colloid. It seems to be completely homogeneous, with no differences in viscosity within a single follicle. There is no
evidence for the existence of vacuoles in the colloid. These
observations confirm the results obtained by Gersh and
Caspersson (’40) and by De Robertis (’41)who used the
f reezing-drying method to prepare their sections. I n addition
observations “in vivo” of the thyroid gland by Vonwiller and
Wigodskaya ( ’34),Hartoch ( ’33), Bucher ( ’38) and Williams
( ’37) had previously emphasized the general optical homogeneity of colloid. Thus evidence from these several sources
makes it appear quite clear that the peripheral colloid vacuoles
described in most preparations made with usual methods, are
artifacts. This conclusion is contrary to the implications of
many histologists working with customary fixatives and stains,
that colloid is of a “solid” nature.
Under certain experimental conditions the viscosity of the
colloid is changed. After administration of potassium iodide
for a few days the viscosity clearly is below the normal, confirming the indirect observations of Loeb et al. (’29). The
viscosity increases however, after prolonged treatment with
the potassium iodide, confirming also the observations of Loeb
(’29) of a secondary increase in the density of the colloid.
The administration of thyrotropic pituitary extract produces
a sharp decrease in the colloid viscosity which is noticed
clearly as soon as 5 hours after the injection.
Changes in the viscosity of the colloid are correlated with
alterations in its proteolytic activity. These observations are
a direct demonstration of the assumption of Loeb (’29) when
he stated that “the softening and solution processes (in K I
administration) are caused by a substance, presumably of an
enzymatic character which is given off by the epithelial cells
when they are in a state of stimulation.” He assumed also
PROTEOLYTIC ENZYME ACTIVITY O F COLLOID
227
that secondarily this enzymatic substance is not secreted and
the colloid becomes solid.
The work of Lerman and Salter ('34)was the first that suggested the possibility that colloid might be reabsorbed from
the follicle after enzymatic hydrolysis. They report some
experiments in which extracts of thyroid tissue were used
t o synthesize thyroid hormone in vitro and they suggested
the possible existence of an enzymatic mechanism which could
catalyze either protein formation or protein destruction according to thermodynamic conditions. It is necessary to
emphasize that the results of Lerman and Salter were obtained with extracts of the whole gland while in this paper
enzymatic activity of isolated colloid is reported.
The same mechanism was invoked by Gersh and Caspersson
('40) as the only one capable of accounting for the great
variability of colloid protein and organic iodine in normal
and experimental animals. It was difficult otherwise to understand how a molecule with such a large molecular weight as
thyroglobulin could pass with such rapidity through the cells
lining the follicle. The existence of this mechanism now is
greatly strengthened by the observations reported in this
paper.
The presence of such proteolytic enzyme activity in the
colloid was clearly established. The fluctuations in the amount
of enzymatic activity, depending on experimental conditions,
support the concept that in the reabsorption of colloid a
proteolytic enzyme is secreted by the thyroid cells which
hydrolizes the large protein molecule of tliyroglobulin. The
reference to one proteolytic enzyme is a convenience; the
existence of more than one enzyme is not excluded by the
methods employed. Results cited by Lerman and Salter
indicate that the enzyme or enzyme system is probably nonspecific since proteases have been detected in extracts from
other organs. The results obtained by the injection of indicators permit us t o affirm that these fluctuations in the activity
of the proteolytic enzyme in the experimental animals are
probably not due to changes in the pH of the colloid. Theoreti-
228
E. DE ROBERTIS
cally, it seems possible that the proteolytic enzyme is also
able to synthesize the thyroglobulin and that a balanced equilibrium may exist in the colloid between those two reactions.
I n the colloid of follicles activated by the pituitary extract
this equilibrium is shifted further toward the hydrolysis side
and the total protein concentration diminishes. The latter is
established by the measurements of Gersh and Caspersson
for the guinea pig. I n the colloid of follicles of rats treated
with potassium iodide, the equilibrium is first in the same
direction (primary activation) ; later, the direction is shifted
in the opposite way (secondary period). The latter stage of
increased protein content in the colloid has been described by
Cersh and Caspersson for the guinea pig. It seems also possible that a similar enzymatic mechanism may exist in the
secretory cells of the thyroid gland. A better knowledge of
the factors that regulate the equilibrium of these enzymatic
reactions in colloid and cells may throw light on the secretory
mechanism of the thyroid gland in normal, experimental, and
pa thological states.
CONCLUSION
1. By microdissection methods colloid was extracted from
single follicles.
2. The colloid is an optically homogeneous, highly viscous,
refractile fluid. Its viscosity diminishes after the injection
of thyrotropic pituitary extract and after a short period of
feeding with potassium iodide. After a longer period of iodide
administration the viscosity increases.
3. The pH of the colloid, determined by the microinjection
of indicators, is 6.6 0.2; that in the perifollicular tissue:
7.3 I+ 0.2. No marked changes in pH were found after the
administration of potassium iodide or thyrotropic pituitary
extract.
4. Diffusion of the indicator in the follicle takes place in
30 to 90 seconds; it is reabsorbed in 3 to 15 minutes.
5. I n the colloid obtained from single follicles is present
a proteolytic enzyme which digests a gelatin substrate.
PROTEOLYTIC ENZYME ACTIVITY OF COLLOID
229
6. This proteolytic enzyme increases in activity in the acid
range and diminishes in the alkaline. Within the physiological
pH range, the activity increases after injection of thyrotropic
pituitary extract and after the administration of potassium
iodide for a short time. After treatment with iodide for
longer time, the activity decreases below the normal level.
7. The demonstration of this proteolytic enzyme, and the
fluctuations in its activity in experimental conditions, support
the concept that an enzymatic mechanism is involved in the
hydrolysis of the colloid protein and the subsequent reabsorption of the products of hydrolysis.
I wish t o acknowledge my indebtedness to Dr. I. Gersh f o r
the suggestion of the problem and for his advice and criticism,
to Dr. H. Jensen of the Squibb Institute for Medical Research
for the thyrotropic pituitary extract, and to this laboratory
for the facilities and courtesies I have received.
LITERATURE CITED
BUCHEB,0. 1938 Untersuchungen iiber den Einfluss verschiedener Fixationsmittel auf das Verhalten des Schilddriisenkolloids. Zeit. f. Zell. u.
mikr. Anat., Bd. 28, S. 359-381.
DE ROBERTIS,
E. 1941 The intracellular colloid of the normal and activated
thyroid gland of the rat studied by the freezing-drying method. Am.
J. Anat., vol. 68, pp. 317-337.
GERSR, I., AND T. CASPF~SSON1940 Total protein and organic iodine in the
colloid and cells of single follicles of the thyroid gland. Anat. Rce.,
V O ~ . 73, pp. 303-319.
HARWCH,
W. 1933 Mikroskopische Beobaclitungen a n lebeiiden Organen.
Speicheldriise und Schilddriise. Klin. Wochenschr., Bd. 12, R. 942-944.
LERMAN,J., AND W. T. SALTER 1936 The bohavior of natural and artificial
thyroid protein. Trans. Am. Ass. for Study of Goiter, pp. 143-154.
LOER,L. 1929 The structural changes which take place in the thyroid glands
of guinea pigs during the process of compensating hypertrophy under
the influence of iodine administration. Endocrinology, vol. 13, pp.
49-62.
PIPKFORD,
G. E., AND F. DORRIS1934 Micromethods for the detection of
proteases and amylases. Science, vol. 80, pp. 317-318.
VONWILLEB, v., AND R. WIGODSKAYA 1934 etudes sur 1eS barrisres histohbmatiques. 11. La thyrboscopie. Observation de la glande thyro’ids, de
ses Blbments histologiques, de ses vaisseaux sanguins et de son produit
de sBcr6tion sur l’aninial vivant. Bull. d. Histol. appl., T. 11,pp. 20-31.
230
E. DE ROBERTIS
J. T.,AND A. N. RICHARDS 1924-1925
Observations on the composition
of glomerular urine, with particular reference to the problem of
reabsorption in the renal tubules. Am. J. Physiol., vol. 71, pp. 209-227.
WILLIAMS,R. G. 1937 Microscopic studies of living thyroid follicles implanted
in transparent chambers installed in the rabbit’s ear. Am. J. Anat.,
V O ~ . 62, pp. 1-29.
WEARN,
PLATE 1
EXPLANATION OF FIGURES
1 Proteolytic activity faintly positive ( & ) of colloid from a rat fed with
potassium iodide during 45 days (pH 7). The clear ring and the central clear
spot observed in this picture are due to folding of the gelatine.
2 id. positive (+) from a normal rat (pH 6.8).
3 id. strongly positive (++) from a rat injected 10 hours before with 10 g.p.
units of thyrotropic pituitary extract (pH 7).
4 id. strongly positive (++) of a rat. fed on K I for 12 days (pH 7).
5 id. strongly positive (++) of a normal rat with buffer pH 2.
I n all cases magnification X 60.
PROTEOLYTIC EKZYME ACTIVITY OF COLLOID
IC. DE ROBERTIS
231
PLATE I
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